The research conducted in this laboratory centers on the molecular mechanism underlying the establishment of neuronal connections during development. We have chosen to focus on the layer-specific targeting of R7 neurons in the Drosophila visual system because (a) it is essential for visual function, and (b) R7 neurons are amenable to genetic manipulation. To understand the logic of R7 target selection, we first characterize the developmental processes. Second, to resolve the complex cellular interactions involved, we genetically ablated the cells of interest and examine the effects on R7 target selection. Finally, developmental mosaic analyses were performed on mutants with R7 targeting defects. We have previously identified N-cadherin (Ncad) and receptor tyrosine phosphatase LAR for their requirement for R7 target selection (Lee et al., 2001). Mosaic analysis showed that Ncad and LAR are required cell-autonomously in R7 neurons for selecting proper target layer. Removing Ncad or LAR in single R7 neurons cause R7 axons mistargeting to R8-recipient layer. To understand how Ncad regulates R7 target selection, we have developed a genetic method to analyze single Ncad mutant R7 axons during development. These analyses reveal that R7 target selection proceeds in three consecutive stages, each of which involves different cellular and molecular interactions. During development, R8, R7 and laminal interneurons (LNs) innervate medullar neuropil in a sequential manner. R8s differentiate first and project their axons into the medulla. Approximately 12 hours later, R7 axons project, along the R8 axon shafts, into the medulla, followed shortly by the LNs. The first stage of R7 target selection involves R7 growth cones defasciculating from R8 axons, thereby allowing R7 growth cones to advance further into deeper medulla. The separation of R7 and R8 growth cones requires LNs whose axons follow R7 and project into the region between R8 and R7 growth cones. Genetic ablation of LNs using an eye-specific allele of hedgehog mutants (hh1) blocks the separation of R7 and R8 growth cones. Although the interaction between LNs and R7 (or R8) appear to be critical, the molecular nature of these afferent-afferent interactions is currently unknown. In the second stage, the R7 growth cones enter the target layer likely by recognizing environmental cues provided by their target neurons. This process requires Ncad function. Removing Ncad in single R7 neurons causes R7 growth cones stop short at the intermediate layer (M4-5) instead of entering the presumptive R7-recipient layer (M6). R7 target neurons sprout dendritic processes in this layer and they also express Ncad. In vitro, Ncad is capable of mediating homophilic interaction. We favor the view that Ncad provides adhesive interaction between R7 growth cones and their targets. Once R7 growth cones reach the presumptive R7-recipient layer, they stop and undergo conformational change, from a spear-like structure into an expanded conformation. This morphological change signifies the beginning of the third stage, the stabilization phase. In LAR mutants, R7 axons project into the correct layer at earlier stages but later retract back to the R8-recipient layer, suggesting that LAR is required for stabilizing the interactions between R7 and their targets (Clandinin et al., 2001). In vertebrates, LAR has been suggested to up-regulate N-cadherin mediated homophilic adhesion by dephosphorylating b-catenin (Kypta et al., 1996; Brady-Kalnay et al., 1998). In this system, LAR might stabilize R7-target interaction by up-regulating Ncad activity. Our analyses reveal that proper R7 target selection depends on a series of complex cellular and molecular interactions. These involved both afferent-afferent interactions as well as afferent-target interactions, executed in a temporally and spatially controlled fashion. At the molecular level, Ncad-based adhesion system appears to play a key role in R7 target selection. The targeting specificity, we believed, is achieved by (a) differential modulation of Ncad adhesive activity in different growth cones and (b) combinatorial use of Ncad and Ncad2 isoforms as well as other surface receptors.